The present invention relates to a novel monoclonal antibody against human von Willebrand factor, which causes no bleeding episodes in a medicinally effective dose to exhibit its antithrombotic action. The present invention also relates to a hybridoma which produces the foregoing monoclonal antibody, and an antithrombotic agent containing the foregoing monoclonal antibody as an active ingredient.
When a subendothelium is exposed due to an injury of vessel walls in a living body, platelets flowing through the bloodstream immediately adhere to the subendothelim. This triggers a series of platelet activation processes including platelet aggregation and release of intracellular granules, after which thrombus is formed, and thus bleeding is arrested. Accordingly, thrombus formation is necessary and indispensable for the physiological hemostatic mechanism. However, on the other hand, the thrombus causes thrombotic diseases such as myocardial infarction, angina pectoris, cerebral infarction, and cerebral thrombosis which become to hold higher ranks of the cause of death in proportion to the aging of society. Such a situation is recognized as a serious problem.
Many antithrombotic agents have been hitherto developed in order to cure and prevent the thrombotic diseases. However, problems to be solved remain in that many of the conventional antithrombotic agents still have low curative effectiveness in clinical application, they have low specificity to thrombus, and they cause hemorrhagic tendency as a side effect. One of the causes of such circumstances is considered as follows. Namely, almost all of the antithrombotic agents are designed only for the purpose of inhibiting the platelet-activating process. A method for measuring platelet aggregation in vitro, which provides an index of the activity, is insufficient to reflect the complicated thrombus formation process in vivo.
Thrombus formation proceeds in accordance with specific binding between glycoprotein on platelet membrane and subendothelium or proteins in plasma. Especially, glycoprotein IIb/IIIa (hereinafter abbreviated as xe2x80x9cGPIIb/IIIaxe2x80x9d) on platelet membrane functions as a receptor for fibrinogen in the final stage of the thrombus formation. Accordingly, it is expected that GPIIb/IIIa-antagonists may be used as a potent antithrombotic agent. The fibrinogen-binding site on GPIIb/IIIa includes an RGD primary sequence of amino acids. As a result of synthesis and evaluation of many RGD derivatives, it has been confirmed that GPIIb/IIIa antagonist exhibits the antithrombotic effect by strongly inhibiting the platelet aggregation, according to an animal models in vivo and clinical investigations (Thrombosis and Haemostasis, vol. 69, p. 560, 1993). However, a problem emerges in that GPIIb/IIIa antagonists simultaneously suppress the normal hemostatic mechanism, and hence the hemorrhagic tendency as a side effect appears more strongly as compared with the conventional antithrombotic agents (The Lancet, vol. 343, p. 881, 1994; The New England Journal of Medicine, vol. 330, p. 956, 1994).
On the other hand, those known as important proteins which function at the early stage of thrombus formation include glycoprotein Ib on platelet membrane (hereinafter abbreviated as xe2x80x9cGPIbxe2x80x9d) and von Willebrand factor in blood plasma (hereinafter abbreviated as xe2x80x9cvWFxe2x80x9d). Hemorrhagic lesions associated with occurrence of qualitative and quantitative change in vWF include von Willebrand disease (hereinafter referred to as xe2x80x9cvWDxe2x80x9d). A clinical knowledge has been obtained that serious bleeding scarcely occurs in vWD patients as compared with patients of thrombasthenia (hemorrhagic disease due to deficiency of GPIIb/IIIa). Therefore, a possibility is conceived that powerful antithrombotic action may be exhibited without involving the hemorrhagic tendency by inhibiting the interaction between GPIb and vWF. However, only a monoclonal antibody and a low molecular weight compound ATA (Aurin Tricarboxylic Acid; Blood, vol. 72, p. 1898, 1988) have been known as substances to specifically inhibit the interaction between GPIb and vWF. Any antithrombotic action of the anti-GPIb monoclonal antibody in vivo has not been confirmed. Instead, side effects are emphasized in that the anti-GPIb monoclonal antibody causes thrombocytopenia, and it prolongs the bleeding time (Blood, vol. 70, 344a, 1987; Jpn. J. Clin. Pathol., vol. 40, p. 266, 1992). Further, it has been reported for those which antagonize vWF that ATA described above and a mouse anti-swine vWF monoclonal antibody BB3-BD5 exhibit antithrombotic efficacies in an in vivo experiment with animals (Circulation, vol. 81, p. 1106, 1990). However, side effects cannot be neglected in the case of both ATA and BB3-BD5. Namely, ATA exhibits the antithrombotic action by inhibiting the interaction between GPIb and vWF, while ATA simultaneously involves completely opposite side effects such that it enhances platelet aggregation and release reaction caused by the aid of collagen, arachidonic acid, A23187, PAF, and TXA2 (Thrombosis and Haemostasis, vol. 68, p. 189, 1992). On the other hand, BB3-BD5 exhibits a strong hemorrhagic tendency in its antithrombotic dose (Proc. Natl. Acad. Sci. USA, vol. 84, p. 8100, 1987; SURGERY, vol. 112, p. 433, 1992).
As described above, there is a dilemma in the existing antithrombotic agents in that the antithrombotic action as an medicinal effect cannot be separated from the hemorrhagic tendency as a side effect (there is no difference between the medicinally effective amount and the amount to cause the side effect).
Recently, shear stress-induced platelet aggregation (hereinafter abbreviated as xe2x80x9cSIPAxe2x80x9d) attracts attention, as closely related to thrombus formation in a pathological state. The vascular diameter is small, and the bloodstream has a large velocity in arteriosclerosis lesions and small arteries. Therefore, a high shear stress occurs in such regions due to the interaction between vessel wall and blood. In such a situation, vWF in blood is activated, and its tertiary structure is changed. As a result, vWF plays a crucial role in thrombus formation. Namely, the following process is known. Firstly, vWF existing on subendothelium binds to GPIb on platelet membrane, and thus platelets adhere to vessel wall. Secondly, vWF existing in blood plasma cross-links glycoprotein IIb/IIIa on platelet membrane, and thus the platelet aggregation reaction is allowed to proceed. Consequently, thrombus formation finally occurs.
It is generally known that an antibiotic ristocetin or a snake venom botrocetin allows vWF to cause a change in tertiary structure in vitro, equivalent to the change under a high shear stress. Namely, in the presence of ristocetin or botrocetin, vWF acquires the binding ability to GPIb. Methods for measuring the physiological activity of vWF in vitro by utilizing the foregoing characteristic include ristocetin-induced platelet aggregation (hereinafter abbreviated as xe2x80x9cRIPAxe2x80x9d) and botrocetin-induced platelet aggregation (hereinafter referred to as xe2x80x9cBIPAxe2x80x9d), as well as a method for measuring binding of vWF to GPIb in the presence of ristocetin or botrocetin. The foregoing methods are widely utilized. Owing to the progress of experimental techniques, an apparatus has been also developed, in which SIPA is measured in vitro by actually applying a shear stress. It is considered that an identical domain on vWF involved in the binding to GPIb in any of the reactions.
Several antibodies against vWF, which inhibit the activity of vWF in vitro, have been hitherto obtained. However, many of them are inferior in reaction specificity, and almost all of them do not inhibit the botrocetin-dependent reaction, even though they inhibit the ristocetin-dependent reaction. As described above, it is considered that the GPIb-binding site on vWF induced by ristocetin is homologous to that induced by botrocetin. Therefore, the foregoing antibodies possibly recognize the binding site on vWF for ristocetin or botrocetin. Strictly speaking, it is possible to say that they do not inhibit the physiological activity of vWF, and hence they have low reaction specificities. In such circumstances, it has been reported that two antibodies, i.e., NMC-4 produced by Fujimura et al. (J. Nara Med. Assoc., vol. 36, p. 662, 1985) and RFF-VIIIRAG:1 produced by Tuddenham et al., inhibit in vitro the reaction depending on both of ristocetin and botrocetin (Blood, vol. 17, No. 1, p. 113, 1991).
It has been reported that epitopes for the two antibodies exist in the GPIb-binding site of the vWF molecule, and they are located between 449th and 728th amino acid residues of an amino acid sequence of the vWF molecule. Further, binding of iodine-labeled NMC-4 to vWF is partially inhibited by RFF-VIIIRAG:1. According to this fact, it is considered that the both epitopes are located at positions considerably close to one another. Moreover, RFF-VIIRAG:1 inhibits BIPA only partially, while NMC-4 completely inhibits BIPA. For this reason, studies have been diligently made in the scientific field of vWF by using NMC-4, and certain results have been obtained. Among animals other than human, NMC-4 has its reactivity only with rat vWF.
When a monoclonal antibody against human vWF is prepared in order to obtain information on the GPIb-binding site of human vWF, or in order to use the monoclonal antibody as a preventive agent and a therapeutic agent against diseases relevant to vWF, it is considered to be desirable to prepare the monoclonal antibody as one having high specificity to human vWF.
On the other hand, when a new medicine is developed in an ordinary manner, it is unallowable to perform any test with human without previously performing a test with animals. When a test is performed in relation to physiological activities of vWF and anti-vWF monoclonal antibodies in vivo, it is necessary to use a monoclonal antibody which makes it possible to perform a test with animals, i.e., a monoclonal antibody simultaneously having reactivity with vWF of an animal other than human. By the way, GPIIb/IIIa antagonists, which strongly suppresses human platelet aggregation by the aid of fibrinogen, are not effective on rat (Thrombosis and Haemostasis, vol. 70, p. 531, 1993). Further, rat does not cause ristocetin-induced aggregation. According to these facts, it is generally considered that the mechanism of thrombus formation greatly differs between rat and human. Therefore, it is almost meaningless to evaluate the antithrombotic action of any anti-vWF antibody by using rat. On the contrary, in the case of guinea pig, platelet aggregation is suppressed by GPIIb/IIIa antagonists. Further, ristocetin-induced aggregation is also induced in the same manner as human. Accordingly, it is considered that guinea pig is most suitable as an animal thrombus model for in vivo experiments when the antithrombotic action is evaluated.
According to the foregoing viewpoints, any of a monoclonal antibody having reactivity with only human vWF, and a monoclonal antibody having reactivity with both human vWF and guinea pig vWF is useful. However, such anti-human vWF monoclonal antibodies are not known.
Further, an anti-human vWF monoclonal antibody, which has been confirmed to have antithrombotic action in vivo, is not known.
The present invention provides, in part, a pharmaceutical composition having antithrombotic efficacy containing a pharmaceutically acceptable carrier and a monoclonal antibody having the following properties:
(a) the monoclonal antibody binds to human von Willebrand Factor; and (b) the monoclonal antibody inhibits binding between a monoclonal antibody produced by hybridoma and human von Willebrand Factor, wherein the hybridoma is selected from the group consisting of FERM BP-5248 (AJvW-2). FERM BP-5250 (AJvW-4) or a variant of the hybridoma.
The present invention has been made taking the foregoing viewpoints into consideration, an object of which is to provide monoclonal antibodies against human von Willebrand factor, especially a monoclonal antibody having reactivity with only human vWF, and a monoclonal antibody having reactivity with human vWF as well as guinea pig vWF, which do not express bleeding action in an medicinally effective dose sufficient to express antithrombotic action, hybridomas for producing the foregoing monoclonal antibodies, and an antithrombotic agent containing, as an active ingredient, any one of the foregoing monoclonal antibodies.
The present inventors have succeeded in obtaining a monoclonal antibody having reactivity with human von Willebrand factor and having action to inhibit RIPA, BIPA, and SIPA of human platelet, by immunizing a mouse with human vWF, and fusing spleen cells of the immunized mouse with mouse myeloma cells to prepare a hybridoma. Further, the present inventors have found out that the monoclonal antibody exhibits strong antithrombotic action without involving bleeding in an in vivo thrombosis model. Thus the present invention has been completed.
Namely, the present invention lies in an antithrombotic agent comprising, as an active ingredient, a monoclonal antibody which has reactivity with human von Willebrand factor, which has action to inhibit RIPA (ristocetin-induced platelet aggregation), BIPA (botrocetin-induced platelet aggregation), and SIPA (shear stress-induced platelet aggregation) of human platelet, and which does not express bleeding action in an medicinally effective dose to exhibit antithrombotic action.
In another aspect, the present invention provides a monoclonal antibody having the following properties:
(a) the monoclonal antibody has reactivity with human von Willebrand factor;
(b) the monoclonal antibody inhibits RIPA (ristocetin-induced platelet aggregation), BIPA (botrocetin-induced platelet aggregation), and SIPA (shear stress-induced platelet aggregation) of human platelet;
(c) the monoclonal antibody inhibits RIPA (ristocetin-induced platelet aggregation) and BIPA (botrocetin-induced platelet aggregation) of guinea pig platelet; and
(d) the monoclonal antibody exhibits strong antithrombotic action in vivo in guinea pig, but it does not cause bleeding.
In still another aspect, the present invention provides a monoclonal antibody having the following properties:
(A) the monoclonal antibody has reaction specificity to human von Willebrand factor;
(B) the monoclonal antibody inhibits RIPA (ristocetin-induced platelet aggregation), BIPA (botrocetin-induced platelet aggregation), and SIPA (shear stress-induced platelet aggregation) of human platelet; and
(C) the monoclonal antibody does not react with von Willebrand factors of rat, guinea pig, and rabbit.
In still another aspect, the present invention provides a hybridoma for producing the monoclonal antibody having the foregoing properties, formed by fusion between spleen cell of a mouse immunized with von Willebrand factor and Sp2/0-Ag14 mouse myeloma cell.
The present invention will be explained in detail below.
 less than 1 greater than  Monoclonal Antibody of the Present Invention
A first embodiment of the monoclonal antibody of the present invention (hereinafter referred to as xe2x80x9cfirst monoclonal antibodyxe2x80x9d) lies in a monoclonal antibody having the following properties:
(a) the monoclonal antibody has reactivity with human von Willebrand factor;
(b) the monoclonal antibody inhibits RIPA (ristocetin-induced platelet aggregation), BIPA (botrocetin-induced platelet aggregation), and SIPA (shear stress-induced platelet aggregation) of human platelet;
(c) the monoclonal antibody inhibits RIPA (ristocetin-induced platelet aggregation) and BIPA (botrocetin-induced platelet aggregation) of guinea pig platelet; and
(d) the monoclonal antibody exhibits strong antithrombotic action in vivo in guinea pig, but it does not cause bleeding.
A specified embodiment of the monoclonal antibody described above is exemplified by a monoclonal antibody further having the following properties in addition to the foregoing properties:
(e) the monoclonal antibody inhibits BIPA (botrocetin-induced platelet aggregation) of rat platelet; and
(f) the monoclonal antibody inhibits BIPA (botrocetin-induced platelet aggregation) of rabbit platelet.
Namely, the first monoclonal antibody of the present invention has high reaction specificity in that it is reactive with human vWF, it has high affinity thereto, and it strongly inhibits any of RIPA, BIPA, and SIPA in vitro. On the other hand, the first monoclonal antibody of the present invention inhibits at least RIPA and BIPA of guinea pig. A monoclonal antibody obtained in Examples described later on further inhibits BIPA of rat and rabbit in vitro. According to an experiment of single intravenous administration to guinea pig, the monoclonal antibody inhibits RIPA and BIPA ex vivo without affecting hematological parameters and coagulation parameters at all. The monoclonal antibody prolongs the time required for femoral artery obstruction in a photochemically reaction-induced thrombosis model based on the use of guinea pig, and it prolongs the time required for obstruction in an arteriovenous shunt formation model. Moreover, when the monoclonal antibody is used in its medicinally effective dose, its effect continues for a long period of time without expressing elongation of bleeding time.
No monoclonal antibody having the properties as described above has been hitherto known. The first monoclonal antibody of the present invention is a novel monoclonal antibody. The first monoclonal antibody of the present invention is clearly different in epitope from NMC-4 described above not only in that it reacts with animal vWF but also in that it does not inhibit binding of NMC-4 to vWF at all (see Examples described later on). The fact that the monoclonal antibody of the present invention has strongly suppressed thrombus formation without involving the bleeding tendency in an in vivo thrombosis model strongly suggest the possibility that the monoclonal antibody of the present invention can be also utilized as an ideal therapeutic agent for thrombotic diseases. The monoclonal antibody of the present invention is not only novel but also industrially applicable.
Namely, the first monoclonal antibody of the present invention is not only useful to specify the GPIb-binding site of vWF. But the first monoclonal antibody of the present invention is also expected to be used as means for analyzing distribution and existing forms of vWF in vivo, and researching the cause of vWD (von Willebrand disease), and to be utilized as a preventive agent and a therapeutic agent effective on thrombotic diseases. Further, the first monoclonal antibody of the present invention can be preferably used for in vivo experiments based on the use of guinea pig when the antithrombotic action is evaluated.
A second embodiment of the monoclonal antibody of the present invention (hereinafter referred to as xe2x80x9csecond monoclonal antibodyxe2x80x9d) is a monoclonal antibody having the following properties:
(A) the monoclonal antibody has reaction specificity to human von Willebrand factor;
(B) the monoclonal antibody inhibits RIPA (ristocetin-induced platelet aggregation), BIPA (botrocetin-induced platelet aggregation), and SIPA (shear stress-induced platelet aggregation) of human platelet; and
(C) the monoclonal antibody does not react with von Willebrand factors of rat, guinea pig, and rabbit.
Namely, the second monoclonal antibody of the present invention is reactive with human vWF, and it has high affinity thereto. Further, the second monoclonal antibody strongly inhibits any of RIPA, BIPA, and SIPA in vitro. Besides, the second monoclonal antibody does not react with any of vWF""s of rat, guinea pig, and rabbit. In view of these points, the second monoclonal antibody has specificity much higher than that of NMC-4.
No monoclonal antibody having the properties as described above has been also hitherto known. The second monoclonal antibody of the present invention is a novel monoclonal antibody. The second monoclonal antibody is clearly different in epitope from NMC-4 described above not only in that it does not react with rat vWF but also in that it does not inhibit binding of NMC-4 to vWF at all (see Examples described later on). According to the fact that the monoclonal antibody of the present invention does not react with vWF""s of those other than human, for example, vWF of rat, it is assumed that the monoclonal antibody of the present invention recognizes a special antigenic determinant specific to human, the antigenic determinant having been not conserved during the process of evolution. This fact is considered to support the high specificity of the monoclonal antibody of the present invention. The monoclonal antibody of the present invention is not only novel but also industrially applicable.
The second monoclonal antibody specifically and strongly inhibits binding between human vWF and GPIb on platelet membrane. Accordingly, the second monoclonal antibody can be utilized as means for specifying the GPIb-binding site of human vWF, analyzing distribution and existing forms of human vWF in vivo, and researching the cause of vWD (von Willebrand disease), in the same manner as the first monoclonal antibody. No in vivo thrombus formation-suppressing experiment has been performed based on the use of animal, because the second monoclonal antibody does not react with vWF""s of animals other than human. However, as demonstrated in Examples described later on, an epitope for the second monoclonal antibody to recognize vWF is located in the vicinity of an epitope recognized by the first monoclonal antibody. Accordingly, the second monoclonal antibody highly possibly recognizes the same epitope as that recognized by the first monoclonal antibody. Therefore, it is assumed that the second monoclonal antibody has an effect equivalent to that of the first monoclonal antibody in vivo. The second monoclonal antibody is expected to be utilized as a preventive agent and a therapeutic agent effective on thrombotic diseases.
The first and second monoclonal antibodies also have action to inhibit shear stress-induced platelet adhesion (hereinafter referred to as xe2x80x9cSIPAdxe2x80x9d) of human platelet. SIPAd also relates to thrombus formation in a pathological state. According to an experiment based on the use of normal human blood, it has been confirmed that the first and second monoclonal antibodies inhibit SIPAd in a dose-dependent manner. Such inhibition has not been observed for GIIb/IIIa antagonists which are expected to be used as antithrombotic agents at present.
A third embodiment of the monoclonal antibody of the present invention is a monoclonal antibody which has reactivity with human vWF, and which has action to inhibit binding between the first or second monoclonal antibody and vWF factor when the third monoclonal antibody is allowed to co-exist with the first or second monoclonal antibody. As demonstrated in Examples described later on, one of the first and second monoclonal antibodies mutually inhibits binding of the other to vWF, using epitopes located closely near to one another or using an identical epitope. Further, the first monoclonal antibody has strongly suppressed thrombus formation without accompanying the hemorrhagic tendency in an in vivo thrombus model. According to these facts, the properties possessed by the first and second monoclonal antibodies that the antibodies inhibit RIPA, BIPA, and SIPA, and they exhibit antithrombotic action, but they do not cause bleeding, are considered to originate from the epitope or opitopes recognized by the antibodies. Therefore, it is considered that the monoclonal antibody, which has the action to inhibit binding between vWF factor and the first and second monoclonal antibodies, can be also as an active ingredient of the antithrombotic agent of the present invention.
The monoclonal antibody having the properties described above can be used as a pharmaceutical. The pharmaceutical specifically includes, for example, an antithrombotic agent as described later on.
 less than 2 greater than  Production of Hybridoma and Monoclonal Antibody of the Present Invention
The monoclonal antibody of the present invention is obtained by performing cell fusion between antibody-producing cells of an animal immunized with human vWF and myeloma cells to form hybridomas, cloning a hybridoma capable of producing a monoclonal antibody having reaction specificity to human vWF and inhibiting RIPA, BIPA, and SIPA of human platelet, and culturing the hybridoma or a variant thereof.
Each type of the monoclonal antibodies is obtained as follows. Namely, the first monoclonal antibody is obtained by cloning a hybridoma capable of producing a monoclonal antibody which inhibits RIPA and BIPA of guinea pig platelet, and culturing the hybridoma or a variant thereof. The second monoclonal antibody is obtained by cloning a hybridoma capable of producing a monoclonal antibody which does not react with vWF""s of rat, guinea pig, and rabbit, and culturing the hybridoma or a variant thereof.
The hybridoma can be prepared in accordance with a method of Kxc3x6hler and Milstein (Nature, pp. 495-492, 1975). A method for preparing hybridomas, and a method for selecting a hybridoma capable of producing an objective monoclonal antibody will be explained below.
Antibody-producing cells are obtained by immunizing an animal, for example, Balb/c mouse with human vWF, and preparing, from the animal, antibody-producing cells such as spleen cells, lymph node cells, and peripheral blood. Human vWF can be obtained by purification from human blood plasma by means of, for example, gel filtration.
The antibody-producing cells are collected from the animal immunized with human vWF to perform cell fusion with myeloma cells. Cell strains originating from various mammals can be utilized as the myeloma cells to be used for cell fusion. However, it is preferable to use a cell strain originating from an animal of the same species as that of the animal used to prepare the antibody-producing cells. In order to distinguish fused cells from unfused cells after the cell fusion, it is preferable to use a myeloma cell strain having a marker so that unfused myeloma cells cannot survive, and only hybridomas can proliferate. For example, a hybridoma, which is formed by cell fusion between a myeloma cell resistant to 8-azaguanine and an antibody-producing cell as a normal cell, is capable of proliferation in a medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine, while the myeloma cell resistant to 8-azaguanine dies in the HAT medium, and the normal antibody-producing cell cannot be cultured for a long period. Therefore, only the hybridoma can be selectively cultured (Science, vol. 145, p. 709, 1964). It is preferable to use, as the myeloma cell, a strain which does not secrete inherent immunoglobulin, from a viewpoint that the objective antibody is easily obtained from a culture supernatant of the hybridoma.
Cell fusion is performed, for example, as follows. Spleen cells of a mouse immunized with human vWF are mixed with mouse myeloma cells, for example, Sp2/0-Ag14 (8-azaguanine resistant, IgG-non-secreting) in the logarithmic growth phase so that the ratio of the spleen cells to the myeloma cells is about 10:1 to 1:1. After centrifugation, a residual precipitate is added with polyethylene glycol having an average molecular weight of 1,000 to 6,000 to give a final concentration of 30 to 50% so that the cells are fused. Fusion may be performed by applying an electric pulse to a mixed solution of the cells, in place of the addition of polyethylene glycol.
Cells having been subjected to the fusion treatment are suspended in HAT medium, for example, Dulbeco""s modified Eagle""s minimum essential medium (hereinafter abbreviated as xe2x80x9cDMEM mediumxe2x80x9d) containing hypoxanthine, aminopterin, thymidine, and 10% fetal bovine serum. The suspension is dispensed and poured into a 96-well microtiter plate or the like, and cells are cultured at 37xc2x0 C. in 5% carbon dioxide so that only hybridomas are allowed to glow.
The hybridomas obtained as described above are provided as a mixed culture containing a hybridoma which produces the objective monoclonal antibody, in addition to hybridomas which produce monoclonal antibodies against other proteins contained in the human vWF preparation in a mixed manner, or monoclonal antibodies against sites of human vWF irrelevant to RIPA, BIPA, and SIPA. Accordingly, a strain, which produces the objective monoclonal antibody, is selected from the foregoing hybridomas.
The hybridoma, which produces the monoclonal antibody having reactivity with human vWF, can be selected in accordance with enzyme immunoassay based on the use of human vWF as an antigen. A strain, which produces a monoclonal antibody to inhibit both of RIPA and BIPA mediated by human vWF, is selected by measuring the inhibiting activity on RIPA and BIPA by using a part of the medium in each well.
The hybridoma, which produces the first monoclonal antibody of the present invention, is obtained by selecting a hybridoma which produces a monoclonal antibody that binds to vWF of an animal such as guinea pig, rat, and rabbit, or a monoclonal antibody that inhibits BIPA or RIPA of platelet of an animal as described above, in accordance with an enzyme immunoassay method such as an ELISA (Enzyme-Linked Immunosorbent Assay) method. The hybridoma, which produces the second monoclonal antibody of the present invention, is obtained by selecting a hybridoma which produces a monoclonal antibody that does not exhibit reactivity with vWF of an animal such as rabbit other than human.
After confirmation of the fact that the hybridoma for producing the objective monoclonal antibody is contained in a culture, the culture is transferred to HT medium having the same composition as that of HAT medium except that aminopterin is removed from HAT medium. The hybridoma is further cultured to perform cloning in accordance with, for example, a limiting dilution method.
Thus hybridomas AJvW-1, AJvW-2, AJvW-3, and AJvW-4 have been obtained as demonstrated in Examples described later on. All of them have been deposited on Aug. 24, 1994 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3 Higashi-1-chome, Tsukuba-shi, Ibaraki-ken, Japan) under deposition numbers of FERM P-14486, FERM P-14487, FERM P-14488, and FERM P-14489 respectively in this order, which have been transferred to international deposition based on the Budapest Treaty on Sep. 29, 1995, and deposited under deposition numbers of FERM BP-5247, FERM BP-5248, FERM BP-5249, and FERM BP-5250 respectively in this order. Among the hybridomas, AJvW-2 and AJvW-4 produce the first monoclonal antibody, and AJvW-1 and AJvW-3 produce the second monoclonal antibody.
As demonstrated in Examples described later on, the monoclonal antibodies produced by AJvW-1 and AJvW-3 belong to the subclass IgG2a isotype, the monoclonal antibody produced by AJvW-2 belongs to the subclass IgG1, and the monoclonal antibody produced by AJvW-4 belongs to the subclass IgG2b. NMC-4 belongs to IgG1 as having been hitherto reported.
The monoclonal antibody of the present invention is obtained by culturing, in an appropriate medium or in mouse ascitic fluid, the hybridoma obtained as described above or a variant selected by cloning the hybridoma in accordance with the limiting dilution method, for example, a variant of the hybridoma having high antibody productivity. Alternatively, the monoclonal antibody of the present invention is also obtained by isolating a gene concerning antibody production from the obtained hybridoma, incorporating the gene into an expression vector, introducing an obtained vector into a microorganism such as Escherichia coli, and cultivating an obtained antibody-producing microorganism. The hybridoma includes AJvW-1, AJvW-2, AJvW-3, AJvW-4 described above, and variants thereof.
The medium for culturing the hybridoma includes, for example, a medium based on DMEM medium and further containing fetal bovine serum, L-glutamine, glucose, sodium pyruvate, 2-mercaptoethanol, and an antibiotic (for example, penicillin G, streptomycin, and gentamicin). The hybridoma of the present invention is usually cultured in the medium at 37xc2x0 C. for 2 to 4 days with a gas phase comprising 5% carbon dioxide and 95% air. Alternatively, the hybridoma is cultured for about 10 to 15 days in an abdominal cavity of BALB/c mouse pretreated with 2,6,10,14-tetramethylpentadecane (for example, Pristane (trade name) produced by Sigma). Thus the monoclonal antibody is produced in an amount capable of being subjected to purification.
The monoclonal antibody thus produced can be separated and purified in accordance with an ordinary method adopted for isolation and purification of proteins from culture supernatant or ascitic fluid. Such a method includes, for example, centrifugation, dialysis, salting out based on the use of ammonium sulfate, and column chromatography based on the use of, for example, DEAE-cellulose, hydroxyapatite, protein-A agarose, and protein-G agarose.
 less than 3 greater than  Antithrombotic Agent of the Present Invention
The antithrombotic agent of the present invention contains, as an active ingredient, the monoclonal antibody which has reactivity with human von Willebrand factor, which has action to inhibit RIPA (ristocetin-induced platelet aggregation), BIPA (botrocetin-induced platelet aggregation), and SIPA (shear stress-induced platelet aggregation) of human platelet, and which does not express bleeding action in an medicinally effective dose to exhibit antithrombotic action. Such a monoclonal antibody specifically includes the first monoclonal antibody and the second monoclonal antibody of the present invention. As described above, it is expected that the monoclonal antibody, which has action to inhibit binding between the first and second monoclonal antibodies and vWF factor when the monoclonal antibody is allowed to co-exist with the first and second monoclonal antibodies, also has the same action as those of the first and second monoclonal antibodies, and it is used as an active ingredient of the antithrombotic agent of the present invention.
When the monoclonal antibody originating from mouse is applied as an antithrombotic agent to human, it is desirable that the monoclonal antibody is modified into one of the human type, because of problems of antigenicity and half-life in blood. Variable regions of the antibody can be converted into those of the human type without losing the reaction specificity in accordance with methods described by Jones et al. (Nature, vol. 321, p. 522, 1986) and Queen et al. (Proc. Natl. Acad. Sci. USA, vol. 86, p. 10029, 1989). Recently, the repertoire cloning method described by Winter et al. and Lerner et al. is also available (J. Mol. Biol., vol. 222, p. 581, 1991; Proc. Natl. Acad. Sci. USA, vol. 88, p. 2432, 1991).
Fragments F(abxe2x80x2)2, Fabxe2x80x2, and Fab, which can be obtained by digesting the foregoing monoclonal antibody with a proteolytic enzyme such as trypsin, papain, and pepsin, followed by purification, can be also used as the antithrombotic agent provided that the fragments have properties equivalent to those of the foregoing monoclonal antibody.
The type or form of the antithrombotic agent of the present invention includes, for example, injection, sublingual tablet, endermic poultice, tablet or pill, capsule, granule, syrup, suppository, ointment, and instillation. Among them, injection, sublingual tablet, and endermic poultice are preferred. Depending on the type of the agent, the antithrombotic agent may be blended with pharmaceutically allowable excipients, for example, lactose, potato starch, calcium carbonate, and sodium alginate. In the case of injection, those used as a solvent include, for example, water for injection, physiological saline, and Ringer""s solution. The solvent may be added with a dispersing agent. Further, an antithrombotic component other than the anti-vWF monoclonal antibodies may be used together.
The dose of administration of the antithrombotic agent of the present invention differs depending on, for example, the age and the condition of a patient. However, in general, in the case of intravenous administration, a predetermined effect can be expected by using the antithrombotic agent of the present invention preferably in a range of 0.1 xcexcg/kg to 1000 mg/kg, more preferably 1 xcexcg/kg to 100 mg/kg per one day for an adult, as represented by amount of the monoclonal antibody to serve as an active ingredient.
The antithrombotic agent of the present invention may be applied to general applications concerning antithrombotic agents. Namely, the antithrombotic agent of the present invention may be applied to prevent or treat diseases relevant to platelet adhesion and aggregation. Specifically, for example, the antithrombotic agent of the present invention is effective in the treatment of transient cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, and peripheral arterial occlusive disease, it is effective in the prevention of reocclusion after PTCA and occlusion of coronary artery by-pass graft, and it is effective in the treatment of coronary artery valve replacement and essential thrombocythemia.